Molecular profiling of gene expression is a powerful tool that can be used to stage human tumors and categorize them with respect to projected prognosis and therapeutic responsiveness. This technology has been applied most effectively to leukemias and lymphomas for which it is relatively easy to obtain highly enriched pools of tumor cells. Similar approaches to study solid tumors have been less successful because solid tumor tissue samples contain many non-tumorigenic types of cells, because many of the tumor cells found in them are dead or dying at any given moment, and because of the low abundance emigrating metastatic cells. Current technology to enrich for metastatic ovarian cancer cells from ascites at the time of surgery involves using a combination of centrifugation and positive- and negative-selecting immuno-affinity steps. This cumbersome protocol yields pools of metastatic cells less than 50% pure and viable that are contaminated by non-tumor leukocyte and epithelial cells and dead and dying tumor cells. In this STTR Phase I application, we propose to optimize and validate a novel and rapid cell separation technology for enrichment of viable metastatic tumor cells from the ascites of patients with ovarian cancer. The approach is anticipated to yield a collection of viable tumor cells greater than 99% pure. In parallel, we will also purify tumor-associated leukocytes to enable expression profiling comparisons. This will provide a measure of the efficiency of the enrichment procedure and a proof-of-principle demonstration of the proposed profiling methods. In Phase II, the integrated technologies optimized in this first phase will be translated into broad gene expression studies on ovarian and uterine cancer: We will perform microarray analysis to identify genes up-regulated in metastatic ovarian and uterine tumor cells from blood and ascites in comparison to non-tumorigenic ovarian and uterine epithelial cells and primary (in situ) ovarian and uterine tumor cells. Ultimately, the combination of specific metastatic tumor cell enrichment and correctly targeted molecular profiling will create a technology capable of staging and predicting prognosis and therapeutic responsiveness for multiple types of epithelial tumors.